A variety of ligands for transmembrane receptor tyrosine kinases regulate raf-1 activity including platelet-derived growth factor, colony stimulating factor-1, insulin, fibroblast growth factor and epidermal growth factor (EGF). We have chosen the EGF receptor (EGFR) system for examination of the regulatory phosphorylation site of Raf-1. Incubation of HER 14 cells with EGF leads to a shift in the electrophoretic mobility, which is caused by an increased phosphate incorporation into the raf protein on serine residues. The specific activity of the Raf-1 kinase is stimulated about sixfold under these conditions. Activated Raf-I physically associates with the EGFR upon EGF treatment in a ligand-dependent manner. Polyclonal Raf-I antisera coprecipitate the EGFR, and monoclonal EGFR antibodies coprecipitate about 1% of the Raf-1 population. However, Raf-1 immunoprecipitates from 32P labeled cells show no phosphate incorporation into T-tyrosine after EGF treatment and instead show a twofold elevated phosphoserine content. Downregulation of protein kinase C (PKC) in HER 14 cells with 12-0-tetradeconyl-phorbol-13-acetate does not block the EGF induced shift in the electrophoretic mobility of Raf-1, suggesting that PKC is not essential for raf-I activation in EGF-treated cells. Therefore, this system demonstrates that there must be a non-PKC mediated serine phosphorylation, which activates the Raf-I kinase activity. Candidate kinases which may be responsible for EGF triggered Raf-1 activation include MAP2 and S6 Kinase. We are currently in the process of determining the predominant serine phosphorylation site of Raf-I in EGF stimulated, PKC downregulated HER 14 cells.